1. Field of the Invention
The present invention relates to methods for producing bacteria that contain eukaryotic genes. More specifically, the invention relates to methods for culturing retrovirally-transformed eukaryotic cells under conditions whereby bacteria containing eukaryotic genes subsequently are isolatable from the culture.
2. Description of the Background Art
During the past century, highly pleomorphic bacteria have been isolated from human patients with a variety of illnesses including cancer, acquired immunodeficiency syndrome (AIDS) and Hodgkin's disease.
In the late 19th century, researchers believed that cancer was caused by an infection. But by the 1920's, after numerous microorganisms were isolated and tested for vaccination potential, researchers discovered that the metastasis of cancer can be caused by the spread of cancer cells within the host. Accordingly, the focus of cancer research turned away from isolation of microorganisms.
However, microorganisms continued to be isolated from the blood and tumors of humans and animals with cancer (See Young, Br. Med. J. (1925) 1:60; Nuzum, Surg. Gynecol. Obstet. (1925) 11:343; Glover, Canada Lancet Pract. (1920) 75:92; Glover et al., Canada Lancet Pract. (1926) 66:49; Scott, Northwestern Med. (1925) 24:162; Stearns et al., J. Bacterial (1929) 18:227). These bacteria often showed characteristics of cell wall-deficient bacteria and could be observed in the blood of cancer patients by darkfield microscopy. Cancer appeared to be induced by injecting these bacteria into experimental animals, and some forms of cancer were shown to be prevented by pre-vaccination with killed bacteria isolated from experimental animals affected with the specific cancer.
For example, Diller vaccinated a group of mice with killed bacteria which had been originally isolated from mice with Sarcoma 180; another group of mice was held as unvaccinated controls. All these mice were then challenged with Sarcoma 180. Sixty percent of the bacteria-vaccinated mice rejected the implants after 10 days and lived indefinitely, but all control mice died of their tumors (Ann. N.Y. Acad. Sci. (1970) 174:65). Similarly, Seibert vaccinated young, inbred female mice from a strain of mice having a high incidence of breast cancer with heat-killed bacteria isolated from a mouse with breast cancer of this same strain. These mice showed a statistically significant delay in developing this seemingly inherited breast cancer as compared with unvaccinated female controls (J. Reticuloendothelial Soc. (1977) 21:279).
The bacteria showed a remarkable tendency toward pleomorphism in culture, sometimes appearing as cocci, sometimes as straight or curved rods, sometimes as motile bacilli, and sometimes mimicking fungi by producing pseudohyphae or larger spore sacs. Some stages of the bacteria could be passed through filters designed to hold back all ordinary bacteria. On culturing these filtrates, the original bacteria would regrow.
Later researchers confirmed and extended these findings (See Wuerthle-Caspe et al., Ann. N.Y. Acad. Sci. (1970) 174:636; Alexander-Jackson, Growth (1966) 30:199; Diller et al., Ann. N.Y. Acad. Sci. (1970) 174:655; Seibert et al., N.Y. Acad. Sci., Series II (1972) 34:504; Inoue and Singer, Nature =l (1965) 205:408). When sent to reference laboratories for identification, the organisms were classified as common bacteria such as Staphylococcus or Corynebacterium species. But the long time often required for their primary isolation, their sensitivity to the composition of the media, the fried egg appearance of many of their primary isolates, and their marked pleomorphism in culture suggested that their in vivo forms were that of cell wall-deficient bacteria (Mattman, "Cell Wall Deficient Microorganisms", CRC Press: Philadelphia, 1974).
More recently, similar highly pleomorphic bacteria have been isolated from the blood and urine of AIDS patients. AIDS is a complex disease in which patients infected with the human immunodeficiency virus (HIV) experience depletion of CD-4 positive lymphocytes and suffer from an array of opportunistic infections and unusual malignancies. The progressive loss of CD-4 positive T cells and subsequent clinical deterioration correlate directly with increased levels of HIV DNA. Some investigators have attributed loss of control over HIV expression to a number of co-factors, including a variety of heterologous viruses and mycoplasma (Chowdhury et al., Biochem. Biophys. Res. Commun. (1990) 170:1365). Mycoplasma fermentans, in particular, has been demonstrated in a high percentage of HIV-infected individuals, but the role of the microbe in AIDS is not well defined. Researchers are attempting to link mycoplasma and AIDS (Macon et al., Human Pathology (1993) 24:554; Lo et al., Lancet (1991) 338:1415; Wang et al., Lancet (1992) 340:1312). In addition, virus-like infectious agents (VLIA's) have been isolated from AIDS patients which have been shown to cause a systemic infection. These VLIA's are heterogenous in both size and shape (Lo et al., Am. J. Trop. Med. Hyg. (1989) 41:364) and have been shown to have a well-defined outer limiting membrane but to lack a cell wall (Lo et al., Am. J. Trop. Med. Hyg. (1989) 40:399).
Hodgkin's diseased is yet another malignancy with evidence of infectious causation and perhaps even contagiousness. Cell wall-deficient bacteria have been isolated from patients. Bunting first isolated a certain bacterium from the glands of untreated cases of Hodgkin's disease. The organism was extremely pleomorphic (Bunting, Bull. Johns Hopkins Hosp. (1914) 25:173). Later, Mazet isolated 26 strains from Hodgkin's patients which were also extremely pleomorphic (Mazet, Montpelier Med. 1941 (1941) 316. Cell wall-deficient ("CWD") bacteria are bacteria which are highly pleomorphic, exhibit poorly developed or absent cell walls, and include not only the mycoplasma, or PPLO's, but also L-form bacteria which have the ability to revert to cell wall producing bacteria in culture. Some CWD bacteria produce a protein resembling chorionic gonadotropin hormone, a substance which appears to protect trophoblastic and cancer cells from immune recognition. There is some evidence that a plasmid may be responsible for this property and even that these bacteria may in some manner be intimately associated with retroviruses. (Macomber, Medical Hypothesis (1990) 32:1-9).
Studies of cell wall-deficient bacteria have been hampered by difficulties encountered isolating and culturing them. Specific strains are often difficult to reisolate. In addition, many questions regarding the evolutionary origins of bacteria, cell wall-deficient or not, and their role in human and animal diseases remain unanswered.